Single-nucleotide polymorphism 309T>G in the MDM2 promoter determines functional outcome after stroke

[Background and Purpose] The E3 ubiquitin ligase MDM2 (murine double minute 2) is the main negative regulator of the p53 protein—a key player in neuronal apoptosis after ischemia. A functional single-nucleotide polymorphism in the human MDM2 gene promoter (rs2279744) regulates MDM2 protein expression. We investigated whether the MDM2 SNP309, by controlling p53-mediated apoptosis, determines functional outcome after stroke.[Methods] Primary cortical neurons were subjected to oxygen and glucose deprivation. Mice were subjected to ischemic (transient middle cerebral artery occlusion) or hemorrhagic (collagenase injection) stroke models. Protein and mRNA levels of MDM2 and p53 were measured in both neuronal and brain extracts. The interaction of MDM2 with p53 was disrupted by neuronal treatment with nutlin-3a. siRNA was used to knockdown MDM2 expression. We analyzed the link between the MDM2 SNP309 and functional outcome, measured by the modified Rankin Scale scores, in 2 independent hospital-based stroke cohorts: ischemic stroke cohort (408 patients) and intracerebral hemorrhage cohort (128 patients).[Results] Experimental stroke and oxygen and glucose deprivation induced the expression of MDM2 in the brain and neurons, respectively. Moreover, oxygen and glucose deprivation promoted MDM2 binding with p53 in neurons. Disruption of the MDM2-p53 interaction with nutlin-3a, or MDM2 knockdown by siRNA, triggered p53 accumulation, which increased neuronal susceptibility to oxygen and glucose deprivation-induced apoptosis. Finally, we showed that patients harboring the G allele in the MDM2 promoter had higher MDM2 protein levels and showed better functional outcome after stroke than those harboring the T/T genotype. The T/T genotype was also associated with large infarct volume in ischemic stroke and increased lesion volume in patients with intracerebral hemorrhage.[Conclusions] Our results reveal a novel role for the MDM2-p53 interaction in neuronal apoptosis after ischemia and show that the MDM2 SNP309 determines the functional outcome of patients after stroke.This work was funded by Instituto de Salud Carlos III (PI15/00473, RD16/0019/0018, and RD16/0019/0001), European Regional Development Fund, Ministerio de Economía y Competitividad (SAF2016-78114-R), European Union’s Horizon 2020 Research and Innovation Programme (Grant Agreement 686009), and Junta de Castilla y León (IES007P17). Drs Rodríguez (RD16/0019/0018), Delgado-Esteban (CP0014/00010), and Sobrino (CPII17/00027) are supported by Instituto de Salud Carlos III.Peer reviewe